Schizophrenia (SZ) genome-wide association studies (GWAS) have identified >100 genome-wide significant (GWS) risk loci. However, risk loci typically span 1 gene and multiple GWS index and proxy SNPs in linkage disequilibrium, leaving causal genes/variants largely unknown, which hinders translating GWAS findings into disease biology and drug targets. It is thus imperative to identify at each locus the functional risk variants, the affected risk gene/s and the associated cellular phenotype changes. Most risk variants are noncoding, and likely influence gene expression through modulating chromatin accessibility to transcription factors (TF). Accessible (open) chromatin overlaps with cis-regulatory sequences and is enriched for common disease risk variants. Our pilot study in neurons derived from induced pluripotent stem cells (iPSCs) also showed an enrichment of SZ GWS index and proxy SNPs in open chromatin flanking TF footprints. We hypothesize that many causal variants at SZ loci modulate TF binding in open chromatin, thereby altering transcriptional and neuronal phenotypes relevant to SZ pathophysiology. We present an innovative approach to identify chromatin-modulating variants at each SZ locus, for both GWS index SNPs and proxy variants. We will directly compare the allele-specific effect of a heterozygous SNP on the quantitative measurements of open chromatin (i.e., ASoC) within the same individual, which minimizes experimental variation and increases assay sensitivity, allowing an effective study using a smaller sample size. We have identified a set of 20 super-heterozygous (super-het) subjects with >80% power to detect ASoC at 70 SZ loci. We found that chromatin openness correlated with gene expression changes from iPSC?neurons at loci of interest, and ASoC was prevalent in iPSC-neurons of a single subject. We propose to extend the ASoC assay to the 20 super-het subjects, and pursue three specific aims: (1) We will map open chromatin by ATAC-seq (Assay for Transposase-Accessible Chromatin by sequencing) in pathophysiologically relevant iPSC-derived neuronal stem cells, dopaminergic and glutmatergic neurons, and search for regulatory SZ-risk variants that present ASoC in sequences flanking TF footprints. (2) We will use multiplex CRISPR-Cas9 genome editing to generate pairs of isogenic iPSC lines that differ only for every 6~7 regulatory SZ-risk variants, and identify the gene(s) cis regulated by each variant by comparing expression differences between the pair of isogenic iPSC-derived neuronal cells. (3) We will prioritize synaptic genes cis-regulated by a regulatory SZ-risk variant and generate isogenic iPSCs differing only for the regulatory variant, and compare neuronal morphological, biochemical, and electrophysiological phenotypes. SZ is a devastating disorder afflicting 1% of the population without cure. This project will help move the field beyond SZ GWAS to deciphering causal mechanisms which will aid in the development of more effective treatments, and will also create a rich research resource of iPSCs carrying SZ GWS risk alleles.